Chymotrypsin, histidine function

In the area of catalysis, the esterolysis reactions of imidazole-containing polymers have been investigated in detail as possible models for histidine-containing hydrolytic enzymes such as a-chymotrypsin (77MI11104). Accelerations are observed in the rate of hydrolysis of esters such as 4-nitrophenyl acetate catalyzed by poly(4(5)-vinylimidazole) when compared with that found in the presence of imidazole itself. These results have been explained in terms of a cooperative or bifunctional interaction between neighboring imidazole functions (Scheme 19), although hydrophobic and electrostatic interactions may also contribute to the rate enhancements. Recently these interpretations, particularly that depicted in Scheme 19, have been seriously questioned (see Section 1.11.4.2.2). 

Much information about the relationship between the stracture of a protein and its function has been determined by site-specific mutagenesis, a technique that replaces one amino acid of a protein with another. For example, when Asp 102 of chymotrypsin is replaced with Asn 102, the enzyme s abihty to bind the substrate is unchanged, but its ability to catalyze the reaction decreases to less than 0.05% of the value for the native enzyme. Clearly, Asp 102 must be involved in the catalytic process. We have seen that its role is to position histidine and use its negative charge to stabilize histidine s positive charge. 

Chymotrypsin has a molecular weight of 23,000 and therefore contains nearly 200 amino acid residues. The question has been posed whether there is some other, yet unknown function inherent in the protein molecule apart from that implied in the mechanism suggested here. Our three-step reaction sequence involves an adsorption site with a specific configuration to result in the initial binding of enzyme and substrate. This absorption site must be in the right spatial configuration towards the catalytic groups (serine and histidine). 

Reflect and Apply Explain the function of histidine 57 in the mechanism of chymotrypsin. 

Another potent group of alkylators are the a-haloketones. The classic example is the alkylator TPCK (tosyl-L-phenylalanylchloromethyl ketone) which interacts specifically with one imidazole ring (histidine residue) in the enzyme a-chymotrypsin (see Section 7.2.3). These compounds are significantly more reactive in Sn2 displacements than alkyl halides. For example, nucleophilic attack by iodide will proceed 33,000 times as quickly on chlo-roacetone as on n-propyl chloride (acetone solvent, 50°C). Inductive pull of the carbonyl function would be expected to both increase the electrophilicity of the methylene function, and stabilize the approaching anionic nucleophile. a-Haloacids and amides will exhibit a similar effect, and both iodoacetic acid and iodoacetamide have found use as biochemical probes for the alkylation of purified enzymes. 

Enzymes may use any of the above mentioned modes of catalysis in order to catalyze a particular chemical reaction. For example, the imidazole ring of a histidine residue of the enzyme a-chymotrypsin (Section 4.4) can function as a general-base catalyst, while in the enzyme alkaline phosphatase, the same residue can function as a nucleophilic catalyst. Indeed, enzymes are complex catalysts which employ more than one catalytic parameter during the course of their action. It is by this successful integration of a combination of individual catalytic processes that a rate enhancement as high as 10 " may be achieved. Furthermore, it is this combination of factors which results in a specific catalyst. 

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